1. Field of the Invention
The invention relates to fluorescence correlation spectroscopy, especially to multicolor cross-correlation spectroscopy, in which the invention can be used to detect interactions between molecules where all the molecules have been labeled with fluorophores that can be excited simultaneously with one laser wavelength but have different emission characteristics.
2. Description of the Related Art
Fluorescence Correlation Spectroscopy (FCS) is a technique that can determine the characteristics of molecular processes by measuring fluorescence fluctuations in a small sample volume (typically a confocal volume) that are caused by the molecular processes. Typical related art FCS technology is described by E. L. Elson and D. Magde (Fluorescence Correlation Spectroscopy. I. Conceptual basis and theory. Biopolym. 13:1-27, 1974) and R. Rigler, U. Mets, J. Widengren, and P. Kask (Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background—Analysis of Translational Diffusion. Eur. Biophys. J. 22 (3):169-175, 1993).
FCS uses only one fluorescent label and is limited in its resolution, and FCS can resolve two processes only when their characteristic times are different by at least a factor 1.6 to 2 (see U. Meseth, T. Wohland, R. Rigler, and H. Vogel. Resolution of Fluorescence Correlation Measurements. Biophys. J. 76:1619-1631, 1999).
Fluorescence Cross-correlation Spectroscopy (FCCS) is also a technique that allows the measurement of association events of two differently fluorescently labeled particles by detecting their distinct signals from an observation volume in at least two detectors (see P. Schwille, F J Meyer-Almes, and Rudolf Rigler. Dual-Color Fluorescence Cross-Correlation Spectroscopy for Multicomponent Diffusional Analysis in Solution. Biophys. J. 72 (April):1878-1886, 1997). The detector signals are cross-correlated and conclusions can be drawn about the association/correlation of the two particles. This technique circumvents the resolution limitations of FCS and can measure any kind of association independent of whether the association changes the molecular process sufficiently. For example, the association does not have to change a molecular process (e.g., diffusion) by a factor of 2 to be measured (for diffusion that means a factor 8 in mass change). However, to achieve the excitation of two fluorophores that have emission characteristics that are sufficiently different to allow separate detection of the two fluorophores, FCCS requires the use of two different laser wavelengths and thus the necessitates the alignment of two laser beams to the same spot in a microscope. This procedure is difficult and has blocked the commercial and scientific exploitation of this technique (see M. Rarbach, U. Kettling, A. Koltermann, and M. Eigen, Dual-color fluorescence cross-correlation spectroscopy for monitoring the kinetics of enzyme-catalyzed reactions. Methods 24 (2):104-116, 2001; N. L. Thompson, A. M. Lieto, and N. W. Allen. Recent advances in fluorescence correlation spectroscopy. Current Opinion in Structural Biology 12 (5):634-641, 2002).
Recently, it was shown that FCCS can be performed with a single laser beam when two-photon excitation is used (see K. G. Heinze, A. Koltermann, and P. Schwille, Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis, Proceedings of the National Academy of Sciences of the United States of America; 97 (19):10377-10382, 2000). The costs of the system and problems of finding fluorophores with adequate two-photon absorption cross sections limit this technique (see O. Krichevsky, G. Bonnet, Fluorescence correlation spectroscopy: the technique and its applications, Rep. Prog. Phys. 65, 251-297, 2002).
It has been suggested that fluorophores with large Stokes' shifts can be used for simultaneous excitation with a single laser beam but no appropriate system has been suggested up to now (see, e.g., K. G. Heinze, M. Jahnz, P. Schwille. Triple Color Coincidence Analysis: One Step Further in Following Higher Order Molecular Complex Formation. Biophys. J. 86, 506-516, 2004). While the related art has found that no appropriate dyes have been found that fulfill the condition for single laser line excitation and emission in two different wavelength ranges. However, a single laser line excitation has recently been demonstrated by two-photon excitation, but no single laser line one photon excitation is found. The first demonstration of single laser line one photon excitation for dual color fluorescence cross correlation in an article published by the inventors (see L. C. Hwang and T. Wohland. Dual-Color Fluorescence Cross-Correlation Spectroscopy Using Single Laser Wavelength Excitation, Chem Phys Chem 5, 549-551, 2004).
FCS and FCCS instruments are commercially available (Carl Zeiss and Olympus). However, they use two laser beams for the excitation of their samples. Alternatively, a single laser for two-photon excitation can be used (IR, pulsed laser).
At least two patents (U.S. Pat. Nos. 6,200,818 and 6,582,903) have claimed the excitation of fluorophores with one single wavelength and their detection in different detection channels due to different Stokes shifts of the fluorophores. However, both patents fail to set forth a system fulfilling the conditions for such measurements.
As a result, at least preferred embodiments of the present invention seek to facilitate fluorescence cross-correlation spectroscopy that uses a single laser wavelength.